Biosensors are devices that can automatically analyze large amounts of gene and protein informations or that can analyze the presence or absence and the function of a physiologically active substance in a relatively simple and rapid manner. Thus, biosensors have been actively applied in various fields, including gene and protein research field, medical field, agricultural, food, environmental and chemical industries, etc. For example, a microarray chip, which is a type of biosensor, is fabricated by immobilizing a probe(s) specific for a target polynucleotide(s) on a glass slide, using a microarray equipment. When it is used, the presence or absence of the target polynucleotide in a sample and a mutation in the sequence of the target polynucleotide can be analyzed by amplifying the target polynucleotide using fluorescence-labeled specific primers, hybridizing the amplification product to the microarray chip and analyzing the fluorescence signal with a scanner.
A microfluidics chip is another type of biosensor. When it is used, whether a trace analyte (DNA, RNA, peptide, protein, etc.) reacts in the chip is analyzed while the trace analyte is allowed to flow into the chamber of the chip. When this microfluidics chip is used, whether the analyte reacts in the chip can be determined by detection of an electrical signal in a relatively simple and quick manner compared to the use of the above-described microarray chip. Thus, the microfluidics chip is highly useful in the medical diagnostic field. Particularly, the microfluidics chip is advantageous from the viewpoint of the miniaturization of the system and the convenience of detection, because it can detect the reaction of the analyte using an electrical signal, not a fluorescence signal. However, this microfluidics chip is problematic in terms of the reproducibility of detection due to a buffer that is received in the reaction chamber. It also has a problem in that a sweeping process for applying an alternating current at each frequency is required, making it inconvenient to measure an impedance value, resulting in difficulty in analysis. Thus, in the biosensor field, there is still a need for a novel sensing-based technology that satisfies both specificity and sensitivity while detecting an analyte in a rapid and convenient manner, and a novel sensor based on this technology.
Meanwhile, with the recent development of nanotechnology, technologies based on gold nanoparticles or silver nanoparticles have been developed. For example, Korean Patent No. 10-0981987 discloses a technology that maximizes sensitivity by amplifying a signal through staining of silver nanoparticles when nano-sized arrays, which are difficult to analyze by a conventional fluorescence-based detection method, are analyzed using a scanning tunneling microscope (STM).
In addition, a fluorophore such as Cy3 or Cy5, which is used in the microarray chip as described above, is inconvenient in that it should be previously labeled onto oligonucleotide primers. In addition, it has problems such as poor optical stability and insufficient light intensity. In an attempt to overcome these problems, fluorophores based on clusters of (oligonucleotide-stabilized silver nanoparticles were proposed (Chris I. Richards et al., Oligonucleotide-Stabilized Ag Nanocluster Fluorophores, J. AM. CHEM. SOC. 2008, 130, 5038-5039).
In addition, a technology that uses silver nanoparticle clusters in the labeling of reporter oligonucleotides produced in target-assisted isothermal exponential amplification (TAIEA) was also reported (Yu-Qiang Liu et al., Attomolar Ultrasensitive MicroRNA Detection by DNA-Scaffolded Silver-Nanocluster Probe Based on Isothermal Amplification, Anal. Chem., May 29, 2012, A-E). In this prior technology, if miRNA is present when TAIEA is performed, it is annealed and amplified, and at that time, a reporter oligonucleotide that indicates the amplification and presence of the target miRNA is produced in addition to an oligonucleotide complementary to the target miRNA, and thus the target miRNA is detected by the detection of the labeled reporter oligonucleotide. In this prior technology, in order to solve problems such as the inhibition of amplification, the induction of nonspecific amplification and the problem in detection sensitivity, which occur when the reporter oligonucleotide is labeled with a Cy5 fluorophore, the reporter oligonucleotide is labeled with a cluster of silver nanoparticles.
However, the prior art technologies as described above relate to either amplifying a signal for electron microscopic observation using silver nanoparticles as an electron microscopic dye or labeling an oligonucleotide with a cluster of silver nanoparticles in place of the fluorophore Cy3 or Cy5, and are far from a new base technology that rapidly and conveniently detects either the presence of a target polynucleotide in a sample or a mutation in the polynucleotide while satisfying both the specificity and sensitivity of detection.
Accordingly, the present inventors have conducted extensive studies to solve the above-described problems occurring in the prior art and to develop a novel detection-based technology that rapidly and conveniently detects either the presence of a target polynucleotide in a sample or a mutation in the polynucleotide while satisfying both the specificity and sensitivity of detection. As a result, the present inventors have found that a silver nanocluster probe comprising a silver nanoparticle binding region and a specific nucleotide sequence region that specifically binds to a target polynucleotide can show stronger emission intensity than conventional silver nanoparticle clusters when silver nanoparticles bind to the silver nanoparticle binding region, and light emission from the silver nanocluster probe will decrease or decay when the target polynucleotide binds to the specific nucleotide sequence region, thereby completing the present invention.